To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform ...(TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.
The TinyArray imager, a robust inexpensive portable imaging device, can detect antibodies against SARS-CoV-2 based on coronavirus antigen microarrays.
Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections ...leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (
vanA
,
nuc
, and
mecA
) and Gram-negative bacteria, including ESBLs (
bla
CTX-M-1
and
bla
CTX-M-2
families) and CREs (
bla
OXA-48
and
bla
KPC
), as well as bacterial speciation (
E. coli
and
Klebsiella
spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.
We report a rapid diagnostic platform that integrates novel one-step blood droplet PCR assay and a high throughput droplet counting system to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood.
The alarming rise in urinary tract infection (UTI) antimicrobial resistance has resulted from a combination of high prevalence, low specificity and the lack of a rapid, point-of-care (POC) antibiotic ...susceptibility test (AST), which has led to the overuse/inappropriate use of antibiotics.
This study aimed to evaluate the performance of a rapid POC phenotypic AST device in reporting susceptibility information within 2 h.
Instrument calibration was performed with model bacteria and fluorescent microbeads to determine the dynamic range and limit of detection for quantifying concentrations of bacteria and demonstrate the ability to rapidly differentiate susceptible and resistant model bacteria. We then evaluated 30 presumptive UTI-positive patient urine samples in a clinical pilot study using a panel of 5 common UTI antibiotics plus a growth control and compared our results to the hospital standard of care AST.
Our device was able to robustly detect and quantify bacteria concentrations from 50 to 10
colony-forming units (c.f.u.) ml
. The high sensitivity of this measurement technique enabled the device to differentiate between susceptible and resistant model bacteria with 100 % specificity over a 2 h growth period. In the clinical pilot study, an overall categorical agreement (CA) of 90.7 % was observed (sensitivity=91.4 %, specificity=88.9 %,
=97) with performance for individual drugs ranging from 85 % CA (ceftazidime) to 100 % (nitrofurantoin).
By reducing the typical timeframe for susceptibility testing from 2-3 days to 2 h, our POC phenotypic AST can provide critical information to clinicians prior to the administration of antibiotic therapy.
Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or asparagine tRNAs. Clinical isolates of
with increased rates of error in gene translation (mistranslation) involving ...the indirect tRNA-aminoacylation pathway have increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection. Moreover, despite parenteral administration of kasugamycin being unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to significantly restrict growth of
in mice. These data suggest that pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation.
Rapidly isolating rare targets from larger, clinically relevant fluid volumes remains an unresolved problem in biomedicine and diagnosis. Here, we describe how 3D particle sorting can enrich targets ...at ultralow concentrations over 100-fold within minutes not possible with conventional approaches. Current clinical devices based on biochemical extraction and microfluidic solutions typically require high concentrations and/or can only process sub-milliliter volumes in time. In a proof-of-concept application, we isolated bacteria from whole blood as demanded for rapid sepsis diagnosis where minimal numbers of bacteria need to be found in a 1-10 mL blood sample. After sample encapsulation in droplets and target enrichment with the 3D particle sorter within a few minutes, downstream analyses were able to identify bacteria and test for antibiotic susceptibility, information which is critical for successful treatment of bloodstream infections.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Yersinia pestis, the causative agent of plague, is a highly pathogenic gram negative bacterium that attenuates the host immune response by translocating effector proteins, known as Yops, directly ...from the bacterial cytoplasm into the eukaryotic cell cytoplasm using a Type III secretion system. The Type III secretion system uses a polymerized needle to translocate Yops into the eukaryotic cell. This self-polymerizing needle is composed of YscF. YscF has been tested as a possible vaccine alternative to other subunit vaccines. Wild type YscF has been previously shown to provide a low amount of protection against challenge with Y. pestis. The focus of this work was to improve YscF as a vaccine candidate. Major epitopes in YscF were mapped to the N-terminus and C-terminus of the protein. The major epitope of YscF was predicted to lie within the fully polymerized needle, obscured from view at the tip of the needle by LcrV, suggesting that the major epitope of YscF is not surface accessible to antibodies, a possible reason for the low level of protection seen in YscF immunization studies. One of the minor epitopes is accessible to antibodies on the surface of the polymerized needle. To drive the antibody response towards the minor epitope, a YscF mutant missing its N-terminus was analyzed. Truncated YscF (trYscF) is not protective. High proinflammatory cytokine levels, were detected in pre-infection sera from mice vaccinated with trYscF. Cytokine levels from trYscF-vaccinated mice were higher than in pre-infection mouse sera from wtYscF vaccinated mice. YscF may have a concealed pathogen associated marker (PAMP) that is exposed in trYscF. Monocytes produced significantly higher amounts of proinflammatory cytokines when treated with trYscF compared to cells treated with wild type YscF. Macrophages produced a significantly higher amount of TNF-α when stimulated with trYscF compared to wild type YscF. NF-κB was found to be activated in cells treated with trYscF and wt YscF. Results demonstrate that YscF represents a new class of pathogen associated molecular patterns.
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